PTH Measurement in Clinical Laboratories

Abstract

In this chapter, we will start with a review of the methodological evolution of the clinical parathyroid hormone (PTH) assays, follow with a detailed discussion of clinical utility, analytical and clinical performances of the current second and third generation assays, their drawbacks and the efforts taken collaboratively by academia and industry to harmonize the PTH assays. Next, we will focus on the profiling of various forms of circulating PTH in healthy and diseases by LC-MS/MS-based analysis, which greatly contribute to the advancement of our understanding in the structure/function and pathophysiology of PTH over the past three decades. Finally, we will comment on the remaining challenges of the present PTH assays for patient management and point to the future research and development needs to meet the unmet medical needs in managing patients with hyperparathyroidism and chronic kidney diseases–mineral and bone disorder (CKD-MBD).

1. Introduction

Calcium and phosphate are critical to skeletal mineralization; while ionized calcium is essential for neuromuscular function and serves as a signaling molecule to communicate and drive intracellular processes. Although, only about 1% of total body calcium and 15% of total body phosphorus is in circulation, the ionized fractions of circulating calcium and phosphate are tightly regulated by the interplay of several hormones to keep their status of homeostasis in response to environmental cues and the physiological needs [1].

PTH and 1,25-dihydroxy vitamin D are the major regulators of calcium metabolism, while PTH and EGF 23 and its cofactor, klotho, work concertedly to control renal excretion of phosphorus and maintain phosphate balance. The complex system is at play to keep these hormones in check in the healthy, while this intricate control mechanism is disrupted in diseases due to the hormone excess/deficiency or loss of the metabolite feedback control such as in patients with parathyroid gland dysfunction or chronic kidney diseases. Therefore, timely and accurately assessing, monitoring, and profiling of these hormones and the important metabolites is essential for the clinicians to understand the degree of calcium and phosphate imbalance when they evaluate the related disorders such as hypoparathyroidism, various forms of hyperparathyroidism, and chronic kidney disease-induced mineral and bone disorder (CKD-MBD) [2, 3].

PTH measurement has been used in the diagnosis and treatment of disorders of calcium/phosphate metabolism because of its predominant role in maintaining the circulating ionized calcium within a very tight concentration range, and in regulating the urinary excretion of phosphorus. PTH measurement is a valuable tool for diagnosing primary and secondary hyperparathyroidism (SHPT). It is also used as a surrogate biomarker to guide the management strategies for CKD patients presenting with systemic mineral and bone disorders (CKD-MBD) and monitoring its progression.

The presence of various circulating forms of PTH and its metabolites, the inter-assay variability and the presence of many variables from sample collection to the test reporting pose significant challenges for accurate PTH quantitation in clinical laboratories and the interpretation of PTH results by clinicians.

2. Evolution of PTH assays

Circulating PTH is a heterogeneous population consisting of full-length PTH (84 amino acids, with a molecular of ~9500 Da) and various sizes of proteolytic C-terminal, N-terminal, and mid-molecule metabolites [4]. In healthy individuals, predominant C-terminal PTH fragments typically started at amino acid position 34, 37, 38, or 45 [5]; a subtype of C-PTH, known as non-1-84 or PTH (7-84), usually starts at amino acid position 4, 7, 8, 10, or 15 with the major fragment presumably starting at position 7 [6, 7]. Full-length PTH (1-84) and N-terminal PTH fragments have very short half-lives (2–4 min), while the C-terminal PTH fragments have a half-life of several hours and even longer in CKD patients with decreased renal clearance [8]. The half-life of PTH (7-84) fragments is longer, ranging from 8.1 to 24.0 min [9]. It was shown that PTH (7-84) fragments are released from the parathyroid gland directly in healthy individuals, but proportionally increase relative to total circulating PTH due to bioaccumulation in patients with CKD [10].

The first C-terminal PTH radioimmunoassay was described by Berson et al. [11]; this and the subsequent first-generation PTH assays employed a single polyclonal antibody against epitopes that were located within the C-terminal part of PTH and thus detected both PTH (1-84) and all C-PTH fragments. It was found that PTH (1-84) and C-terminal PTH fragments accounted for 20% and 80% of the circulating PTH respectively in healthy adults when measured by the first-generation PTH assay [12]. Meanwhile, in CKD patients, the proportion of measured C-terminal PTH fragments increased to 95% of circulating PTH [13]. The first generation PTH RIA assay is time-consuming and lacks specificity, especially in CKD patients and thus was totally replaced by more specific second-generation sandwich assays.

Nichols Diagnostics developed a two-site immunoradiometric assay (IRMA) for measuring PTH in 1987; this assay uses a capture antibody directed against the 39-84 C-terminal epitope region and a signaling antibody directed towards the 13−4 N-terminal epitope region to form antibody-PTH-antibody complex and thus greatly improved sensitivity and specificity of the PTH quantitation [14].

Subsequently, non-radioactive labeling assay formats (ELISA, chemiluminescent, and electrochemiluminescent methods) were brought forth and operated in automated immunoanalyzers in clinical laboratories of all sizes, which became and still remain to be the most widely-used PTH assays to date. These second-generation assays were collectively known as “intact” PTH assays, for it was thought that they measure only the full-length PTH (1-84). However, it was uncovered later that the “intact” PTH assays still cross-react with PTH (7-84) fragments (ranging from 50% to 100%), and thus overestimated PTH concentration in CKD patients [15].

To improve the diagnostic accuracy, the first third-generation “biointact,” (also known as “bioactive” or “whole”) PTH (1-84) assay, was advanced by Scantibodies Laboratories and became available as an exoteric testing service since 1999. It is an immunochemiluminometric assay with a signaling antibody directed against the epitope within the first 4 amino acids at the very N-terminus of full-length PTH [16]. More recently, non-radioactive automated and FDA-cleared third-generation assays were marketed by several manufacturers including DiaSorin, Tosoh, Fujirebio, Roche, and bioM´erieux [17]. Since third-generation assays have higher specificity to PTH (1-84) and won’t cross-react with C-PTH, the measuring values are approximately 50–70% of those measured by the second generation PTH assay in patients with CKD and approximately 15% lower than those in persons without CKD. Because second generations assays have been used for decades and are still widely in use, this inter-generation assay difference complicates the test interpretation and the adoption of the new assays. It was expected that the use of the third-generation assays can resolve the issue of cross-reactivity with non-functional PTH fragments (i.e., PTH 7-84). However, subsequent studies revealed the complexity of PTH physiology that was undetected by the older PTH assays. In addition to the full-length PTH (1-84), the third-generation “biointact” PTH also reacted with a new form of N-terminal PTH (N-PTH) that is not recognized by most second-generation PTH assays. Further investigation showed that posttranslational phosphorylation at serine position 17 of the PTH (1-84) molecule prevent (or reduces the binding affinity of) the signaling antibody in most second-generation assays from binding to its epitope in the phosphorylated N-PTH. N-PTH accounts for 4–8% versus 15% of circulating PTH measured by the third generation PTH assays in healthy versus in patients with CKD [16, 18].

3. Unresolved problems with PTH assays

3.1 Inter-assay variability

Currently, automated second-generation and third-generation PTH assays are employed in clinical laboratories for PTH measurement. Current second-generation assays provide convenient and relatively reliable methods (intra-assay imprecision <10 %) for PTH measurement [19]. However, different PTH assays from various assay manufacturers measure different types and amounts of the circulating PTH forms depending on the specificity of the antibodies used to construct the assay, which led to great inter-assay variability and inconsistent results among the PTH measurements when the now-obsolete Allegro PTH intact assay served as the reference [20, 21, 22]. In a more recent study, a performance comparison among six currently-existing second-generation assays was made. Imprecision was evaluated using three concentrations of commercial quality control materials, while inter-assay variability was assessed by paired comparisons using 203 serum and 193 EDTA plasma samples from healthy individuals. The results showed that the imprecision (i.e., total coefficients of variation) were between 1.1% and 10.9% and there was a good correlation for all methods overall but the considerable bias was observed between methods, the Bland-Altman plots revealed that the between assay differences were between +1.6% to −36.3%, influenced by both assays and sample types used [23].

The results of third-generation PTH assays are approximately 50–70% of those measured by the intact PTH assay in patients with CKD and approximately 15% lower

than those in persons without CKD [16, 18]. The automated third-generation assays are calibrated against the WHO 95/646 Standard and therefore displayed significantly improved inter-method agreements [24, 25]. However, the incompatibility in measurement to that of the widely-used second generation assays affect the interpretations of the method validation and may contribute to its slow adaptation to clinical laboratories in general.

3.2 Aggravating heterogeneity of circulating PTH in the disease states

The pathological changes in calcium and phosphate status and the progressive loss of feedback control in the calcium and phosphate regulatory system in hemodialyzed patients further exacerbate the problem of assay variability. A systematic performance evaluation of 15 commercial immunoassays with 47 serum pools from dialysis patients, reported by Souberbielle et al. [21] in 2006, showed great inter-assay variability among the tested PTH assays, moreover, the discrepancies of measured values in some assays compared to the then “gold-standard” Allegro PTH intact assay are unacceptable, and may cause patient harm when the discrepant results were used to make therapeutic decisions. This raised the alarm in the dialysis community to question the reliability of the PTH testing and resulted in many more investigations on these critical issues. A recent position paper issued by the IFCC Committee for Bone Metabolism tabulated 23 major assay comparison studies using samples from CKD or hemodialysis patients (published between 2005 and 2018) [17]. The results reaffirmed that existing between-method differences in PTH measurements did not improve much and likely have treatment implications.

As described earlier, third-generation assays cross-react with a phosphorylated form N-PTH was overproduced in some patients with parathyroid carcinoma and severe primary hyperparathyroidism (PHPT) [26, 27]. In these cases, PTH determination with the third generation assay will have a value greater than the one with the second-generation assay. The inverted third/second PTH ratio is therefore proposed as a screening or monitoring tool for parathyroid cancer [28]. However, the inter-assay variability makes it challenging to define a generally acceptable cutoff for validating the proposed clinical utility unless the problem of analytical variability is effectively addressed.

3.3 Issues of concern related to testing procedures

3.3.1 Pre-analytical phase: sample stability, sample type, and sampling time

The unstable nature of PTH makes it essential to optimize pre-analytical parameters, including specimen type, sampling time, and storage conditions, which have all been thoroughly investigated. After a systematic review conducted under the auspice of IFCC PTH Working Group, the following evidence-based recommendations are made by IFCC: [29, 30].

• For samples collected with EDTA tubes, the plasma must be separated from the cells within 24 hours of venipuncture. Samples should be kept at 4°C and analyzed within 72 hours of venipuncture.

• For serum samples, the serum must be separated from the cells as soon as possible, and PTH is analyzed within 3-4 hours of venipuncture.

• Central venous PTH concentrations were higher compared to peripheral venous PTH concentration, therefore, in patients undergoing hemodialysis or parathyroidectomy, if the blood samples were collected via central line or central vein; the collected tube, as well as the test report, should explicitly state the collection site and whether they are peripheral or central venous concentrations.

• PTH follows a circadian rhythm, exhibiting a nocturnal peak, a mid-morning nadir, and a smaller afternoon peak. Therefore, it is suggested that samples for PTH measurement should be collected between 10:00 and 16:00, preferably in the morning with an overnight fast. Other known biological variations include the fact that PTH level increases with age and BMI, and is generally higher in African Americans than in Caucasians.

PTH has longer stability in EDTA tube at room temperature than in serum tube, thus delayed centrifugation to allow blood clotting is not needed; however, it is important in clinical practice that PTH measurement be accompanied by a concomitant calcium value. Since calcium (and bone-alkaline phosphatase) cannot be measured in EDTA plasma, PTH and calcium are to be measured in the same serum tube and therefore may be a preferred option for practical reasons. In two recent reports, PTH values obtained from the rapid serum tubes were found to be decreased compared to those from the serum separator tubes (SSTs) [31, 32].

So far, there is no reported data on the comparison of relative PTH stability using second-generation versus third-generation assays. Such study is valuable in providing further insight into the ex vivo stability/vulnerability of each type of PTH molecule.

3.3.2 Analytical phase: heterophilic antibodies interference

All immunometric assays are inherently prone to interference from heterophilic antibodies (i.e., human idiotypic antibodies that interact with assay antibodies raised from animals). Such interference can lead to diagnostic errors and may cause harm to patients as consequence. Assay manufacturers have introduced effective blockers to the assay reagent as a preventive measure against heterophilic antibody interference; however, increasing use of modified monoclonal mouse antibodies as therapeutics in recent years makes heterophilic antibodies interference a special concern in the patients who receive such treatment. Clinicians and laboratorians should keep open communication when the testing results did not match the clinical picture of the patients. Laboratorians should offer adequate confirmatory measures and be able to interpret the investigative results timely and correctly to avoid the spurious results being used to make important clinical decisions for patient management [33].

3.3.3 Post-analytical phase: reference ranges

Since a great inter-assay variability is still present among current commercial PTH assays, there also exists a significant difference for the reference ranges provided by the manufacturers. This makes validation of the PTH assay in use an indispensable but tedious and challenging job. The first and foremost is the selection of the reference population; The eGFR, serum calcium, and 25[OH]D values of the candidate samples should be determined to only include those within reference ranges, especially the 25[OH]D level should be >30 ng/mL [34, 35]. The reference ranges should be established for each sample locally used. If types of collection tubes differ among the collection sites, it is advisable to perform a comparison study to determine if the reference range should be revised to accommodate the difference. In high latitude areas, the effect of seasonal variation in 25[OH]D levels may need to be taken into consideration in designing the validation study for establishing the local reference range for PTH assay.

3.4 Inability to discern oxidized from non-oxidized PTH by current clinical PTH assays

Loss of biological activity of oxidized PTH in vitro was observed as early as 1934, it was later showed that oxidation of two methionine residues at positions 8 and/or 18 within the receptor-binding domain results in the altered three-dimensional conformation of PTH, which in turn results in greatly reduced affinity to PTH receptor [36]. Other in vitro studies also provide evidence that oxidized PTH does not stimulate cAMP-mediated signal after binding to its cellular receptor and thus lost the ability to activate muscle contraction, nor can it stimulate mouse calvarial bone cells to increase alkaline phosphatase activity, which indicates that excessive oxidative stress may disrupt calcium and phosphate homeostasis through oxidation of PTH [37].

There is plenty of experimental evidence to support the increase of oxidative stress in patients with CKD due both to the depletion of anti-oxidants and increase of reactive oxidative species (ROS) production; increasing oxidative stress is also shown to be associated with complications such as hypertension, atherosclerosis, and anemia and therefore may contribute to the accelerated disease progression and mortality in CKD [38]. However, none of the current second-and third-generation assays can discern oxidized from non-oxidized PTH until recently due to the lack of appropriate analytical tools for investigation.

Hocher et al. developed a two-step method to measure the non-oxidized PTH; the oxidized PTH molecules were first removed by an immunoaffinity column with monoclonal antibodies specifically against the oxidized human PTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) fragment, the remaining non-oxidized PTH was then measured by Roche second-generation PTH assay. The method was applied to analyze 17 hemodialyzed samples and revealed a substantial but variable portion (70−90%) of the total PTH was in oxidized form for all samples tested [39]. These tools enable the researchers to assess iPTH, non-oxidized PTH and oxidized PTH simultaneously using the same parameters in clinical research settings to address the clinical association of oxidized PTH with the progression of CKD [40, 41]. However, the results from these studies are not conclusive.

The inherent problems of the analytical procedure are a concern. First, the ex vivo oxidation after sample collection cannot be ruled out; second, the recovery of non-oxidized PTH after the immunoaffinity column removal of oxidized PTH is unknown in clinical samples, and most troubling of all, iPTH assays has not been standardized yet, it is known that some of the iPTH assays use a signaling antibody that is raised against the epitope close to the second oxidation site (methionine at position 18) in PTH; the avidity of such signaling antibody may be changed by the oxidation of PTH. Therefore, replacing second-generation intact PTH with a more specific third-generation PTH (1-84) assay, introducing the spiked internal control to calculate recovery after column treatment, and devising standard operating procedures to minimize and evaluate the extent of ex vivo oxidation will improve the reliability of this assay. For now, the non-oxidized PTH is not ready for clinical use unless all the issues described above are appropriately addressed.

3.5 Clinical implications of the problematic PTH measurements

Lack of a common PTH reference range not merely cause inconvenience, but it also affects results interpretation, and possibly clinical management, especially for monitoring long-term changes of PTH level if patients are not able to use the same health care facilities. More importantly, intraindividual biological variability of PTH is known to increase in hemodialysis patients. The negative impact of PTH assay variability on the management of patients with CKD and hyperparathyroidism is especially troublesome.

3.5.1 Clinical practice guidelines for PTH measurement in CKD-MBD

In 2003, National Kidney Foundation—Kidney Disease Outcomes Quality Initiative (KDOQI) published a guideline that recommended maintaining a target range of 150–300 pg/mL for intact PTH concentrations in stage 5 CKD patients to reduce the mortality related to CKD-MBD. However, the recommendation was based on the comparison of PTH measurements using Allegro iPTH (now obsolete) with the gold standard–bone biopsy, before the problem of inter-assay variability being revealed. A later study showed that iPTH (measured by Immulite DCP assay) levels less than 150 pg/ml for identifying low turnover and greater than 300 pg/ml for high turnover presented a positive predictive value of 83% and 62%, respectively, moreover, in patients achieving the target iPTH levels, 88% had low turnover diseases [42]. The great inter-assay variability is a likely contributing factor to the poor results and indicated the misclassifications may cause harmful clinical outcomes [21].

In one study conducted by the United Kingdom National External Quality Assessment Service (UK-NEQAS), a 4.2-fold difference between highest and lowest measured PTH concentrations were observed using five commonly-used second-generation assays when testing EDTA plasma from 21 hemodialysis patients. In a subsequent study, 98 patient samples were tested by the same iPTH assays to derive assay-specific target values based on Passing and Bablok regression against Roche Elecsys E170 assay which gave the closest results to target values recommended by clinical guidelines. By applying the corrected assay-specific target values, the misclassifications of bone turnover reduced from 53% to 12% [43].

The Kidney Disease Improving Global Outcomes (KDIGO) 2009 Guidelines for the Diagnosis, Evaluation, Prevention and Treatment of CKD-MBD expanded the scope and refined the recommendations to assist clinicians in treating patients with CKD Stages 3–5 who are on dialysis. Aware of the problem of inter-assay variability, this guideline avoids the use of absolute PTH values but suggests hemodialyzed patients maintain PTH levels between two and nine times the upper normal limit (ULN) of the assay used and emphasize on trending the changing pattern rather than the value per se. PTH values above the target suggest high bone turnover bone disease with a specificity of 86%, while PTH levels below the target value suggest low bone turnover with a sensitivity of 66%. In its recent update (July 2017), targets for CKD-MBD biomarkers, including PTH, remain unchanged [44]. As mentioned earlier, 25[OH]D status has a substantial influence on physiological PTH level, but PTH assay reference ranges offered by the vendors might be established with samples of poor 25[OH]D status. Recent studies reported that the upper reference range for PTH established with a reference population with normal GFR and calcium levels, and a 25[OH]D level >20 ng/mL is significantly lower than the reference range provided by the manufacturers [35, 45, 46]. Cavalier et al. found that applying KDIGO guideline PTH target ranges using reference ranges established in a vitamin D replete healthy control population would reduce the percentage of misclassification of bone turnover in dialyzed patients to 16% (versus 36% using vendor-established reference range) [34].

3.5.2 PTH assay incompatibility and the diagnosis of PHPT

Comparison studies compared Nichols iPTH versus Bio-intact PTH assays and Scantibodies Laboratory’s Total versus Whole PTH assays showed high diagnostic sensitivity for both second-and third-generation PTH assays (89−97%), which provided evidence that both types of PTH assays are valuable tools in diagnosing PHPT and provide comparable results [47].

It is essential, as described earlier, to use a vitamin D-replete population to establish the reference range for PTH; the diagnostic accuracy (sensitivity and specificity) for PHPT was shown to be improved in a vitamin D-replete population [46, 48]. However, there is debate over using sufficiency level (30 ng/mL) or insufficiency level (20 ng/mL) as the threshold. So far, there are still no established reference intervals for second-and third-generation PTH assays using large vitamin D-replete population cohorts; subjects with hypercalcemia and a PTH persistently within the upper reference range should be considered “asymptomatic” PHPT and closely monitored [49].

3.5.3 Assay-dependent rate change in intra-operative PTH testing

Because PTH (1-84) has a very short half-life (~5 min), measuring PTH concentrations during parathyroidectomy can inform surgeons whether the pathological parathyroid tissue has been removed completely. A 50% decline of PTH compared with the preoperative level is commonly used to define treatment success. Since second-generation assays cross-react with C-terminal fragments with a longer half-life, it is reasonable to think third-generation assays will perform better for intraoperative PTH monitoring. Studies so far did not demonstrate that third-generation assays have a better performance for ioPTH in patients with PHPT. But a more rapid rate of PTH drop was observed in the third-generation assays. In surgery performed in patients with SHPT, it takes time for PTH concentrations to drop below the 50% cutoff after removal of the last hyperplastic gland using second-generation assays. More studies are needed to determine if third-generation assays offer a superior clinical utility for ioPTH monitoring, especially in patients with SHPT [50].

In addition, PTH measurement is used 20 min after thyroidectomy to determine if intensive calcium monitoring is needed (when the PTH level is >15 pg/mL using iPTH assays) and 4 hours after to predict postoperative hypocalcemia [51, 52]. There is no published study comparing the performance of different-generation PTH assays for this particular purpose, although one can argue that third-generation PTH measurement may better reflect the biological activity of parathyroid glands.

4. Toward standardization of PTH assays

There is a desperate need to reduce the significant inter-assay variability of PTH measurement that confounds the test interpretation and may affect clinical decision-making and cause patient harm. Assay standardization is therefore urgently required to improve the long-standing troubling situation in clinical PTH testing. Hormonal immunoassay standardization is inherently challenging because of the low circulating concentration, protein instability, and the differences in antibody-antigen complex formations; the presence of various PTH fragments and their different distributions in response to calcium, 25(OH) D status, and other effectors present additional hurdles to overcome.

International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), equipped with the experience for standardizing thyroid hormone and other protein assays, is undertaking the challenge to improve the PTH measurement in clinical communities. The IFCC Committee on Bone Metabolism has laid out the roadmap in a position statement where they set three major priorities:

• Calibrate all current commercial PTH assays against a recognized International Standard, proposed to be the recombinant human PTH 1-84 standard (NIBSC 95/646) prepared by World Health Organization.

• Facilitate the development of reference measurement procedure (RMP) for PTH (1-84) to enable the true metrological assignment of reference value for PTH primary, secondary and working standards in a network of reference laboratories.

• Design studies to establish common reference intervals for PTH assays [30].

4.1 Assessing assay commutability and establishing reference PTH sample panels

The first task for the IFCC Working Group is to assess the commutability of PTH in a defined matrix, which is to demonstrate experimentally that the standard material and fresh patient specimens exhibit the same analytical response (regression line slope close to 1.0) when measured by two different methods.

A collaborative effort is currently undertaken to develop a protocol for the formal assessment of commutability. Once the commutability of the standard material is determined and deemed acceptable, it will be possible to use RMP to determine the recovery of PTH (1-84) in the appropriate matrices and then certify the values for secondary reference materials and external controls. Once such standards are available, assay manufacturers should use them to calibrate their assays.

In the meantime, the work is also underway to acquire an appropriate panel of plasma and/or serum samples for establishing PTH reference intervals. All the pre-analytical and physiological factors that can contribute to intra- and inter-individual variations as well as to increase inter-assay variability, as discussed in the previous section of this chapter, are to be carefully considered and minimized or ruled out.

One obvious challenge for this endeavor is the lack of consensus on how to define vitamin D sufficiency, insufficiency, and optimal vitamin D levels. The most often used definition of vitamin D sufficiency is the 25[OH]D concentration above which PTH cannot be suppressed further, however, this threshold varies with disease states and is subject to analytical variability of 25-(OH) D assays [39]. Recently, a cross-sectional analysis of 14,289 CKD patients (stages 1−5) and a randomized control trial involving 429 patients with stage 3-4 CKD, showed levels of iPTH was not suppressed until serum 25(OH) D reached 40−50 ng/ml range, therefore, the target 25-(OH) D concentration may need to be raised in CKD patients [53, 54].

4.2 Developing candidate reference PTH measurement procedures for assay standardization

The liquid chromatography-tandem mass spectrometric (LC-MS/MS) method utilizes chromatographical separation and distinct mass/charge ratio of the product ion pairs to provide rigorous physicochemical characterization of target molecules in the biological mixture and therefore is ideal for developing reference measurement procedures for PTH. The technical advances in mass spectrometric analysis in the last decade enable LC-MS/MS to quantify PTH with accuracy and precision comparable to the results obtained with immunoassays in complex matrices, while it is more robust and flexible for identifying and measuring new or modified PTH fragments (i.e., oxidized PTH). Several published LC-MS/MS methods for PTH measurement already exist and can be readily refined and modified to become candidates for RMPs [5, 55].

IFCC working group has conducted a feasibility study as the first step to assess the suitability of a selected LC-MS/MS method as RMP for PTH quantification. In this study, 48 freeze-dried proficiency testing specimens with assigned values sent from UK NEQAS were reconstituted and analyzed by a published LC-MS/MS method used at the Mayo Clinic [56]. Results obtained from LC-MS/MS analysis were in excellent agreement with the target all laboratory trimmed mean used in the UK NEQAS for PTH and thus the feasibility of using the LC-MS/MS method as a candidate reference measurement procedure.

However, the analytical sensitivity of current MS methods still could not match that provided by immunoassays for PTH quantitation; moreover, current methods require proteolytic digestion of PTH before MS analysis, which is time-consuming and can introduce significant procedural variability. Some of the MS methods also include an immunoabsorbent step to select and enrich PTH. The specificity of the antibody used will influence what types of PTH fragments later be analyzed by LC-MS/MS and thus introduce biases to the final results. Therefore, there remain many hurdles to overcome in developing an MS-based RMP for PTH measurement [30].

Moreover, the employment of state-of-art liquid chromatography-high resolution mass spectrometry (LC-HRMS) could potentially profile various PTH fragments in different stages of CKD with a sensitivity comparable to that by immunoassays and thus offer a powerful tool to correlate PTH qualitative and quantitative changes with the progression of CKD [57].

5. Conclusions

Accurate quantitation of circulating PTH is challenging due to the presence of various molecular forms of PTH and its complex physiological interactions with other hormones and its effectors—calcium, and phosphate. PTH assays have been continuously evolving and improving since their debut six decades ago; the effort will continue to refine and adapt to resolve the issues at hand and meet the evolving clinical needs.

To improve the reliability of the PTH testing for diagnosis and monitoring along the pathway of patient management, IFCC has spearheaded an ambitious plan to standardize commercial PTH assays. It will require the collaborative efforts of academics, scientific and clinical communities, assay manufacturers, and the support of other stakeholders to achieve the goals. We can be optimistically hopeful that the communicable PTH reference material, the panels of qualified samples for establishing reference ranges, and the LC-MS/MS-based RMP method(s) will be available in the foreseeable future. Together they will enable calibration of all PTH assays with a single reliable international standard and allow accuracy-based external quality assessment. The better analytical tool can also empower us to gain more insight into the dynamic changes of PTH so we can optimize the testing to be used in the management of CKD to benefit patient care.